Contemporary assays have enabled single molecule detection (Betzig and Chichester (1993) Single Molecules Observed by near-Field Scanning Optical Microscopy. Science 262(5138):1422-1425; Levene et al. (2003) Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299(5607):682-686) have accelerated the sequencing of the human genome (Anonymous (2001) Unsung Heroes. Science 291(5507):1207) and facilitated imaging with extraordinary resolution without labels (Hell S W & Wichmann J (1994) Breaking the Diffraction Resolution Limit by Stimulated-Emission—Stimulated-Emission-Depletion Fluorescence Microscopy. Opt Lett 19(11):780-782). To most closely study an interaction in the natural state, an assay would interrogate the processes (reaction, molecular interaction, protein folding event, etc.) without perturbation. Label-free chemical and biochemical investigations (Liedberg et al. (1995) Biosensing with Surface-Plasmon Resonance—How It All Started. Biosens Bioelectron 10(8):R1-R9; Yu et al. (2014) Shedding new light on lipid functions with CARS and SRS microscopy. Bba-Mol Cell Biol L 1841(8):1120-1129) transduce the desired signal without an exogenous label (fluorescent, radioactive, or otherwise) representing an essential step toward this goal. Many label-free methods require one of the interacting species to be either tethered or immobilized to the sensor surface, introducing a potential perturbation to the natural state of the species (Moreira et al. (2005).
However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (Bornhop et al. (2007) Free-solution, label-free molecular interactions studied by back-scattering interferometry. Science 317(5845):1732-1736). There are other techniques performed in free solution, such as mass spectrometry (MS) (Cubrilovic et al. (2014) Quantifying Protein-Ligand Binding Constants Using Electrospray Ionization Mass Spectrometry: A Systematic Binding Affinity Study of a Series of Hydrophobically Modified Trypsin Inhibitors. J Am Soc Mass Spectr 23(10):1768-1777; Kaltashov et al. (2012) Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics. Biotechnol Adv 30(1):210-222) and nuclear magnetic resonance (NMR) (Hu et al. (2004) The mode of action of centrin—Binding of Ca2+ and a peptide fragment of Karlp to the C-terminal domain. J Biol Chem 279(49):50895-50903; Tzeng and Kalodimos (2011) Protein dynamics and allostery: an NMR view. Curr Opin Struc Biol 21(1):62-67) and the widely used isothermal titration calorimetry (ITC) (Ababou and Ladbury (2007) Survey of the year 2005: literature on applications of isothermal titration calorimetry. Journal of Molecular Recognition 20(1):4-14; Liang, Y. (2006) Applications of isothermal titration calorimetry in protein folding and molecular recognition. J Iran Chem Soc 3(3):209-219). As with NMR, ITC has many advantages, but exhibits modest sensitivity and often requires large sample quantities. Another increasingly popular free-solution approach is micro-scale thermophoresis (MST). Yet, for MST to operate label-free, one of the binding partners must have a significant absorption/fluorescence cross-section (Wienken et al. (2010) Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 1; Zhang et al. (2014) Microscale thermophoresis for the assessment of nuclear protein-binding affinities. Methods Mol Biol 1094:269-276). BSI represents an attractive alternative to these methods because of its high sensitivity, small sample volume requirement, optical simplicity and broad applicability (Baksh et al. (2011) Label-free quantification of membrane-ligand interactions using backscattering interferometry. Nat Biotechnol 29(4):357-360; Kussrow et al. (2012) Interferometric Methods for Label-Free Molecular Interaction Studies. Anal Chem 84(2):779-792; Olmsted et al. (2014) Toward Rapid, High-Sensitivity, Volume-Constrained Biomarker Quantification and Validation using Backscattering Interferometry. Anal Chem 86(15):7566-7574; Saetear et al. (2015) Quantification of Plasmodium-host protein interactions on intact, unmodified erythrocytes by back-scattering interferometry. Malaria J 14). Whereas ITC and MST have well known or established theoretical descriptions, the fundamental mechanistic basis for the signal observed in BSI is less well understood.
Accordingly, there remains a need in the art for systems and methods for free-solution, label-free detection of intermolecular interactions between analytes, preferably with low detection limits and/or low sample volume requirements.